The present invention relates to an easy and quick process for the extraction and purification of Paclitaxel from natural sources. In comparison to the prior processes known in the art, this process is particularly economical because it has a limited number of steps and a reduced amount of losses during the purification.
The first work concerning Paclitaxel, also called Taxol(trademark) or Pacitaxeline(trademark), started in the United States in the 1960""s, when the National Cancer Institute began a program for selecting plant extracts having activities against cancerous tumours, or anti-neoplasic activities.
From 1960 to 1981, more than 110,000 composites were extracted from 35,000 species of plants and were isolated and tested (Blume E. J. Natl. Cancer Inst. 1991; 83: 1054-1056).
Yew is part of the plants that were selected and tested, and the first extract from yew barks (Taxus brevifolia Nutt) coming from the west coast of the United States (Oregon) was obtained by Wani et al (Wani et al, J. Am. Chem. Soc., 1971; 93:2325-2327). The crude extract of these barks demonstrated a cytotoxic activity against leukaemic cells and an inhibiting action against a variety of tumours.
A couple of years later, the active compound of the extract was isolated. This active compound has been given Paclitaxel as generic name, and its molecular structure has been determined by X-ray crystallography and by 1H-NMR spectrum (Wani et al. J. Am. Chem. Soc. 1971; 93:2325-2327).
Since that time, studies on the effect of Paclitaxel towards cancerous tumours in-vitro and in-vivo have been carried out and the positive results that were obtained have lead to classify this active compound as one of the promising drugs for the treatment of ovarian cancer and breast cancer (Rowinski et al. Pharmacol. Ther. 1991; 52: 35-84). By the way, the use of Paclitaxel for the treatment of different cancers has been approved by the Food and Drug Administration since 1992.
The first variety of yew that was used to produce Paclitaxel, was Taxus brevifolia, but other varieties found in different regions of the earth have also been tested. They consist of Taxus baccata, Taxus canadensis, Taxus wallichiana, Taxus yunnanensis, Taxus densiformis, Taxus hicksii, Taxus wardii, Taxus cuspidata, Taxus capitata, Taxus brownii (Miller et al. J. Org. Chem. 1981; 46: 1469; McLaughlin et al. J. Nat. Prod. 1981; 44: 321; Kingston et al. J. Nat. Prod 1982; 45: 466; Senilh et al. J. Nat. Prod. 1984; 47: 131-137; Huang et al. J. Nat. Prod. 1986; 49: 665-669; Fett-Neto et al. Bio/Technology 1992; 10: 1572-1575).
All these species contain Paclitaxel, but in very limited amountsxe2x80x94about 0.0004 to 0.008%xe2x80x94(Kingston, Pharmacol. Ther. 1991; 52: 1-34). This low concentration of Paclitaxel makes its extraction and purification very costly because it takes time and it generally calls for repeated chromatographies.
The low concentration of Paclitaxel in all the varieties of yew produces a huge impact on the environment. To extract 1 kg of Paclitaxel from Taxus brevifolia barks, one needs to cut down around 3000 grown trees to get 10,000 kg of bark. The obtained quantity (1 kg) of Paclitaxel permits to treat about 500 patients but the number of patients suffering from cancer is as high as hundreds of thousands. Replanting the trees will never meet the urgent demand of Paclitaxel for human needs, because of their slow growth (Vidensek et al. J. Nat. Prod. 1990; 53: 1609-1610; Kelsey et al. J. Nat. Prod. 1992; 55: 912-917; Wheeler et al. J. Nat. Prod 1992; 55: 432-440).
Numerous processes for extracting and purifying Paclitaxel have been proposed. For example, Wani et al. (Wani et al. J. Am. Chem. Soc, 1971; 93: 2325-2327) have proposed a process for extraction of Paclitaxel from the barks of yew of the United States"" west coast (Taxus brevifolia) comprising a treatment of the barks with alcohol followed by a few steps of purification by chromatography.
Miller et al. (1981) have extracted Paclitaxel from the Taxus wallichiana Zucc by a the following process:
1) extraction from the plant and concentration of the extract;
2) fat removal by separation of water and hexane;
3) extraction with chloroform and concentration;
4) first purification by chromatography in a first silica column;
5) second purification by chromatography in a second silica column;
6) a first countercurrent distribution;
7) a second countercurrent distribution;
8) preparative HPLC chromatography.
Senilh et al. (1984) have isolated Paclitaxel (or Taxol(trademark) A: 0.0165%), Cephalomannine (or Taxol(trademark) B: 0.0064%) and other compounds from barks of the Taxus baccata by the following process:
1) extraction with alcohol and concentration;
2) separation of water and dichloromethane;
3) filtration chromatography;
4) chromatography in a silica column;
5) alumina chromatography;
6) chromatography in a medium pressure silica column;
7) HPLC chromatography.
For other analog, two or three other column chromatography treatments followed by a preparative HPLC chromatography are necessary.
Another process used by Polysciences Inc. comprises the following steps:
1) dried ground barks are treated with methanol or ethanol and the obtained extract is concentrated to remove the alcohol,
2) the concentrate is then treated with dichloromethane and the obtained solvent extract is concentrated to yield a powder,
3) the powder is dissolved with a mixture of acetone and ligroin (1:1) and filtered to remove insoluble matter,
4) the organic phase which contains Paclitaxel is concentrated, dissolved in 30% of ligroin, and applied to a column of Florisil(copyright),
5) the Paclitaxel fraction from the column is purified by double crystallization,
6) the so obtained crystalline Paclitaxel is subjected to chromatography on a silica column. The Paclitaxel is separated from the other taxanes (related analogs, cephalomannine, etc.) in this step,
7) the purified Paclitaxel obtained from the previous step is crystallized twice, and
8) unseparated mixtures and mother liquors are recycled through the silica column to obtain additional amounts of pure Paclitaxel.
Of course, there are other processes for purifying Paclitaxel from natural sources, as is explained in the following references:
Kingston et al., who disclose new taxanes obtained from Taxus brevifolia (J.
Nat. Prod. 1982; 45: 466-470); and
Witherup et al., who disclose a method for the separation of Paclitaxel and related compounds from Taxus brevifolia (J. Liq. Chrom. 1989; 12: 2117-2132).
U.S. Pat. No. 5,279,949 issued to Nair in 1994 discloses the use of tissue from ornamental yew (Taxusxc3x97media Hicksii) for Paclitaxel purification. In this patent:
1) fresh needles are extracted with 70% alcohol,
2) the extract is decolorized with charcoal and filtered,
3) the filtered extract is concentrated to remove most of the organic solvent,
4) the aqueous concentrate is centrifuged to separate the solids which contain Paclitaxel,
5) the solids are then subjected to a first normal phase silica chromatography,
6) the obtained crude Paclitaxel fraction is subjected to a second, low pressure silica chromatography; and
7) final purification is carried out in a reverse phase column.
U.S. Pat. No. 5,654,448 issued to Pandey et al., discloses a process for extraction and purification of Paclitaxel from barks of Taxus brevifolia. In this process:
1) the barks are treated thrice with methanol, each extraction being performed over a period of 5 days, the so-obtained extract being then concentrated;
2) the methanol concentrate is separated to obtain methylene chloride and water and the methylene chloride extract is evaporated to dryness, the solid residue containing the Paclitaxel amounting to about 1.8-2.2% w/w;
3) the solid residue is dissolved in acetone and mixed with one volume of hexane to remove polar impurities, and the mixture is evaporated to ⅓ of its volume;
4) the acetone/hexane residue is added drop-wise to hexane (1.5 L-3.0 L of residue for 10-15 L of hexane) to yield a precipitate which is filtered and dried under high vacuum (1 mm to 2 mm) at a temperature of 40xc2x0 C. to yield about 0.5-0.6 kg of a solid residue;
5) the solid residue obtained in the previous step is dissolved in 0,5 L of acetone-methylene chloride to form a 1:9 v/v mixture and subjected chromatography in a silica column; Cephalomannine is co-eluted out with Paclitaxel; the fractions containing Paclitaxel and Cephalomannine are pooled together and rotary evaporated to dryness, the solid residue being a crude mixture of Paclitaxel and Cephalomannine containing from 36 to 40 grams of Paclitaxel (45-55%);
6) a crude mixture (10 g) obtained in the previous step is chemically modified to separate Paclitaxel from Cephalomannine by bromination; the solid residue obtained after bromination reaction having a weight of 13.2 g;
7) the brominated residue is dissolved in acetone/methylene chloride (1:9 v/v) and chromatographically separated over a silica column, the fractions containing Paclitaxel are pooled together and evaporated to dryness; and
8) the solid residue obtained in the previous step is dissolved in acetone and crystallized with an equal volume of n-hexane or other hexanes; and the crystals are washed with cold acetone/hexane, 1/1 v/v solution, filtered, and dried under vacuum at 40xc2x0 C., the so obtained solid crystals weighing 4.84 g and containing  greater than 97% w/w of Paclitaxel as measured by HPLC chromatography.
U.S. Pat. Nos. 5,475,120 and 5,670,673 issued to Rao discloses a process for the isolation of Paclitaxel. In this process:
1) barks, needles, wood, roots, or a combination thereof, are treated with an alcohol and the obtained extract is concentrated under pressure ( less than 35-40xc2x0 C.) to remove most of the alcohol;
2) the concentrate is partitioned with chloroform (or dichloromethane, dichloroethane or trichloroethane), the chloroform extract is concentrated under reduced pressure to form a thick syrup, and the syrup is poured into glass dishes and dried in a vacuum oven ( less than 40xc2x0 C.) to form a powder if barks or wood are used, or needle-shaped crystals if needles are used (from 100 kg of the wood or barks, the yield of extract was 1.5-2.5 kg and from 100 kg of needles, it was 2.4-4.8 kg);
3) chloroform solid extracts (2-2.5 kg) are dissolved in acetonitrile (5 L) and water (5 L) was added; the mixture was then equilibrated with silica gel (2-3 L of slurry); more water (15 L) is added gradually while stirring the mixture; the clear solution is siphoned off and the thick slurry of silica gel which is now impregnated with the sample, is transferred onto the top of a stainless steel column; fractions containing Paclitaxel and various important Paclitaxel analogs, are set aside in a hood where slow evaporation of the solvent is carried out; crystals forming after in 1 to 2 days and the process is allowed to continue for 8-10 days; and
4) the so-obtained crude crystals which have less than 5% of Cephalomannine, are crystallized twice with a acetone/ligroin mixture and by using charcoal, thereby yielding 41 g of pure Paclitaxel, viz. a yield of 0.04%, the above yield being obtained with 100 kg of bark; alternatively, the crude crystals are decolorized by passing a solution in chloroform through a short column of silica or Florisil(copyright). Another alternative method for removing the Cephalomannine contaminant consists of using ozone. Washing of the column with a mixture of 1-2% methanol in chloroform gave a bulk of Paclitaxel which can be recovered by concentration and crystallization. The yield is nearly the same as before, 40 g. HPLC chromatography analysis shows that the so recovered Paclitaxel contains less than 0.3% of Cephalomannine.
U.S. Pat. No. 5,969,165 issued to Liu discloses a process for the isolation and purification of Paclitaxel and other related compounds from Taxus canadensis by industrial preparative low pressure chromatography.
In this process, needles and twigs of Taxus canadensis (200 kg) are extracted with 1000 L of methanol at 60xc2x0 C. for 5 hours and then filtered. The raw materials are extracted with 700 L of methanol at 55-60xc2x0 C. for another 4 hours and filtered. The filtrate is combined and mixed with 10 kg of activated carbon (5% w/w). The activated carbon is removed by filtration. The filtrate is then concentrated to approximately 100 L. Then 300 L of water and dichloromethane (1:1) are added. The organic layer is collected and the aqueous solution is extracted two more times with 200 L of dichloromethane. The dichloromethane solution is combined and evaporated to form a slurry and then diluted with 20 L of acetone.
The acetone solution is coated onto 20 kg of Celite(copyright) 545 and dried and then loaded onto the top of three industrial low pressure chromatographic columns (150 times. 15 cm). Each column is packed with 15 kg of an aluminium oxide absorbent. The columns are eluted with a solvent system consisting of a mixture of hexane and acetone under a pressure between 10 and 15 psi with a flow rate of about 150 ml/min.
Fractions containing taxanes are collected and combined and then are concentrated to remove all other solvents. The resulting material is dissolved in methanol and kept at room temperature overnight to yield needle-like crystals. The crystals are filtered and recrystallized from acetone to yield white needle-like crystals identified as 13-acetyl-9-dihydrobaccatin III.
The filtrate is concentrated to dryness. The residue is dissolved in 3 L of acetone. The acetone solution is mixed with 1.5 kg of a polystyrene-divinylbenzene. The solvent is removed by evaporation and the resulting powder is loaded on top of an industrial low-pressure chromatographic column packed with polystyrene-divinylbenzene, and is eluted with 45% acetone in water at a flow rate of 150 ml/min under an operating pressure below 30 psi.
Fractions containing Paclitaxel and Cephalomannine are combined and evaporated to remove most of the acetone, then diluted with deionized water and extracted three times with 2,5 L of dichloromethane. The organic layer is concentrated to dryness and the residue is dissolved in 1 L of methanol.
Approximately 30% (v/v) of water is added to the methanol solution and the mixture is warmed to 60xc2x0 C. for a few minutes, and then kept at room temperature overnight. The crude crystalline solid from the methanol solution is filtered out and dried in a vacuum oven at 70-75xc2x0 C. The solid consists of approximately 70% Paclitaxel and 25% Cephalomannine in a yield of 31 grams for 200 kg of needles and twigs of Taxus canadensis. 
The crude Paclitaxel is dissolved in 200 ml of acetonitrile and diluted with 250 ml of deionised water and loaded to a chromatographic column. The chromatographic column is packed with a polymer resin (Diaion(copyright), which is a macroporous polymethacrylate resin). The column is eluted with a step gradient of 35, 40, 45 and 50% acetonitrile in water.
The obtained fractions containing Paclitaxel or Cephalomannine are separately combined and kept at 5xc2x0 C. until crystallization. Then, Paclitaxel and Cephalomannine crystals are filtered separately and both are recrystallized at 65xc2x0 C. with a mixture of methanol and water.
Paclitaxel is obtained as white needle-like crystals with a purity  greater than 99% at yield of 18.5 g (0.009%). Cephalomannine is obtained with a purity  greater than 98% at a yield of 6.5 g (0.003%).
Thus, review of the known processes for the purification of Paclitaxel as disclosed in the prior art known to the Applicant shows that to obtain a high purity Paclitaxel, one must carry out numerous steps of separation by chromatography and purification by crystallization. This leads to a very high production cost due in particular to the low of Paclitaxel in the different species of Taxus. Moreover, the amount of biomass which can be purified, is very limited because of the small sizes of the chromatographic columns and because of the low yield in Paclitaxel obtained after purification.
A first object of the present invention is to provide a process which permits:
to make easier the obtention of a biomass after extraction of the barks, needles and/or branches of Taxus of different species;
to increase the amount of biomass which is so obtained and has to be purified by chromatography;
to reduce the steps of purification;
to increase the amount of obtained Paclitaxel; and finally
to reduce the production cost to a more economical level.
Another object of the invention is to provide a mixture of Paclitaxel crystals having a high purity.
In accordance with the present invention, the first object mentioned hereinabove is achieved with process for the extraction and purification of Paclitaxel from a natural source of taxanes containing the Paclitaxel to be extracted, which process comprises the following steps:
a) extracting, with an organic solvent, a raw material comprising Paclitaxel from said natural source of taxanes;
b) treating said raw material with a basic medium or an acidic medium to obtain a biomass by precipitation, isolating said biomass and drying it;
c) removing resin and natural pigments contained in the isolated and dried biomass by dissolving said biomass in acetone and then adding thereto at least one non-polar solvent until a Paclitaxel-enriched oily phase is obtained;
d) treating the biomass contained in the Paclitaxel-enriched oily phase recovered in the preceding step with an acidic medium when step (b) was carried out with a basic medium, or with a basic medium when step (b) was carried out with an acidic medium to obtain another biomass by precipitation, isolating said precipitate and drying it;
e) chromatographically purifying at least once a solution of the isolated other biomass obtained in the preceding step in a volatile solvent, and crystallizing at least once the purified solution obtained by chromatography.
In accordance with the invention, the second object mentioned hereinabove is achieved by a mixture of Paclitaxel crystals, which is obtained by the above process and which, after filtration and drying of the crystals consists of:
about 53% of crystals having a purity higher than 99%,
about 22% of crystals having a purity higher than 98%, and
about 23% of crystals having a purity higher than 92%.
The present invention, its advantages and the way it can be reduced to practice will be better understood upon reading the following non-restrictive description.